PERIPHERAL NERVE ISCHEMIA-We will continue our studies on the pathophysiology and treatment of experimental nerve ischemic fiber degeneration (IFD) and demyelination. We will study the molecular pathogenesis of nerve ischemia and reperfusion injury (Specific aim number 1), with particular focus on the roles of: (1) cytokines, (2) reactive oxygen species, (3) caspase-3, and (4) the important role of host defenses. Insights into the molecular pathophysiological chain could lead to expanded options in the prevention and treatment of nerve IFD. We will study the effects of ischemia alone and compare this with the effects of ischemia-reperfusion on nerve IFD and demyelination. We will use immunohistochemical methods to study ICAM expression and the inflammatory response, separately studying infiltration of polymorphonuclear neutrophils and monocytes. The expression pattern of TNF-alpha, IL-1beta, IL-6, and IL-10 mRNA will be measured using competitive RT-PCR, and their cellular localization by immunohistochemistry of the protein and in situ hybridization of the mRNA. These studies will be augmented in rats by selective anti-cytokine therapy and by the use of wild and genetically altered mice, of identical background genes, that overexpress, or do not express (knockout) TNF-alpha. The role of oxidative stress will be evaluated by measurements of endoneurial 4-hydroxynonenal (HNE), lipid hydroperoxides, and reduced glutathione (GSH). Cellular localization of oxidative damage will be sought, with particular focus on the Schwann cell, using immunohistochemical labeling of HNE. Caspase-3 message, protein expression and cellular localization will be evaluated using competitive RT-PCR, immunhistochemistry and in situ hybridization. We will test the hypothesis that reperfusion injury has two effects, augmenting IFD and selectively causing demyelination. We posit that demyelination evolves more slowly, affects a different population of fibers and is mediated by apoptosis (evaluated using light microscopy [TUNEL] staining) and electron microscopy. We will use single teased fiber analysis to categorically separate out axonal degeneration from demyelination. We will then define the therapeutic windows and evaluate the mechanisms of production of hypothermic and antioxidant neuroprotection (Specific aim number 2) from reperfusion injury.